Project summary Pseudomonas aeruginosa (PA) is a versatile opportunistic pathogen that is a leading cause of hospital- acquired infections. PA antibiotic resistance continues to explode, making development of new therapeutic approaches a critical need. One largely unexplored therapeutic venue is the uncommonly large number of sensing systems that PA has evolved. These signal transduction pathways allow PA to rapidly adapt to a wide variety of environments, such as transitioning from swimming to surface-associated states. Through genetic screens, we and others have identified three systems in PA?the type IV pilus (TFP), the Chp chemosensory system (a complex chemosensory system), and the second messenger 3', 5'-cyclic monophosphate (cAMP) and its allosterically regulated protein binding partner, Vfr-- that are required for upregulation of virulence factors upon surface binding. Activation of the Chp system by TFP retraction leads to a cascade of phosphorylation events that leads to the activation of a membrane-bound adenylate cyclase (CyaB), the primary enzymatic source of cAMP. cAMP binds to a transcriptional activator (Vfr) to induce transcription of >200 genes, many of which are involved in virulence in humans and in causing acute lung damage. We have discovered that in response to surface binding and retraction, the TFP functions as a mechanotransducer to activate the Chp phosphorelay, which in turn increases the activity of the transmembrane adenylate cyclase CyaB. Using multiple approaches, we have uncovered interactions between various components of this system that identify two key signal integrating hubs. We propose 3 specific aims to test and further refine our model and that will deepen our understanding of TFP-Chp-CyaB mechanochemical signaling pathway. Aim 1. Test the hypothesis that PilJ serves as a central integrator of MCS by coordinately regulating the mechanical input signal (altered pilin monomers) with activation of the Chp phosphorelay and with activation of CyaB. We will use genetic screens, in vivo assays of physiologic function, in vivo biochemistry, and live cell fluorescence imaging including FRET to define the (A) PilA/PilJ/CyaB/ and (B) PilJ/PilH interaction landscapes. Aim 2. Test the hypothesis that FimV/FimL/PilG hub links TFP function to the Chp/CyaB system. We will (A) define the FimL/PilG interaction landscape and (B) use Phos-tag technology 16 to examine PilG and PilH phosphorylation in vivo during MCS. Aim 3. Define key spatial and temporal properties of TFP-Chp-CyaB mechanochemical signal transduction during biotic biofilm formation on polarized lung epithelial monolayers. We will (A) Determine the contribution of TFP-Chp-CyaB MCS during biotic biofilm formation and (B) Determine the temporal and spatial dynamics of the surface-activated gene expression during biotic biofilm formation.